TLR3 expression was measured through flowcytometry in freshly isolated PBMCs after TLR3 siRNA treatment. Nonspecific binding of antibodies was blocked using Fc block (1 μg/mL, BD Fc block). Freshly isolated cells were incubated with TLR3 antibody or isotype control in flow cytometry staining buffer (1:250) for 30 min at 4 °C. After 3 washes with PBS, cells were incubated with Alexa Fluor 488 conjugated secondary antibodies (Invitrogen) for 15 min. Cells were washed thrice with PBS and subjected to flow-cytometric analysis by using a BD-FACS Caliber system (BD Biosciences, Franklin Lakes, CA, USA).
Fc block
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Australia, France, Uruguay, Canada
The Fc block is a laboratory equipment designed to block Fc receptors. It functions as a tool to prevent non-specific binding of antibodies during various immunological assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (45 mentions)
Lsrfortessa, by BD (32 mentions)
Lsrii flow cytometer, by BD (27 mentions)
Facscalibur, by BD (26 mentions)
Facsdiva software, by BD (25 mentions)
Facsaria 2, by BD (22 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (19 mentions)
Facscanto 2 flow cytometer, by BD (19 mentions)
Lsr 2 flow cytometer, by BD (16 mentions)
Cytofix cytoperm, by BD (16 mentions)
Lsr 2, by BD (15 mentions)
Dnase 1, by Roche (14 mentions)
Dnase 1, by Merck Group (14 mentions)
Accuri c6 flow cytometer, by BD (13 mentions)
Golgiplug, by BD (13 mentions)
Lsrfortessa flow cytometer, by BD (13 mentions)
Software, by FlowJo (12 mentions)
Facscanto, by BD (12 mentions)
Cytofix cytoperm kit, by BD (11 mentions)
Perm wash buffer, by BD (11 mentions)
761 protocols using fc block
1
Measuring TLR3 Expression in PBMCs
2
Distinguishing CD45+ Cells and CTCs via Multimarker Immunofluorescence
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To differentiate CD45+ cells and CTCs in our sample, we performed a live cell stain using direct immunofluorescence (DIF) with an antibody cocktail (Supplementary Table S1 ). The cocktail included 1 uL of PRAME [30 (link)], MCAM [31 (link)], MCSP [32 (link)], c-Kit [33 (link),34 (link)], PD-L1 [4 (link),5 (link)], MART-1 [35 (link)], and CD45, and 0.5 uL of hoechst nuclear stain (20 mM). FC block (BD Bioscience, Franklin Lakes, NJ, USA) was added to the cells at a concentration of 1 µL FC block per 100 µL of blocking solution, and incubated for 15 min at 4 °C. Next, 1 µL of each antibody from Supplementary Table S1 and Hoechst stain (1 ug/mL) were added. Cells were incubated at 4 °C for 20 min in the dark. The cells were washed with 1 mL of staining buffer (0.5% BSA, 0.02% NaN3 in PBS) and spun at 300 g for 5 min. The cells were washed again with 1 mL of staining buffer and then centrifuged at 300 g for 5 min. Staining buffer was gently aspirated off, and the cells were resuspended in 200 µL PBS and plated on the CytoSort array, prepped with Corning Cell-Tak (final array volume 2.2 mL).
3
Comprehensive Immune Cell Profiling in Liver and Blood
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Kupffer cells were washed with PBS/1% BSA/0.1% NA azide (staining buffer), blocked with Fc block (BDbiosciences, San Jose, CA) for 10 min on ice, incubated with anti-human CD45 (BDbiosciences) for 30 min, followed by intra-cellular staining with anti-human CD68 (clone# Ki-M7, AbD serotec, Raleigh, NC) using BD Cytofix/Cytoperm fixation/permeabilization kits (BDbiosciences). Hepatocytes were washed with staining buffer followed by intra-cellular staining with goat anti-human serum albumin (MyBiosource, San Diego, CA). Approximately 5 million events were acquired for each staining. Peripheral blood (1–2 μl) drawn from the vein of the tail was collected in heparin-coated capillaries (Sigma-Aldrich, St Louis, MO), washed twice with PBS, suspended in PBS/1% BSA, blocked with Fc block for 15 minutes at RT and stained with anti-human CD235 (BDbiosciences) and Retic-Count (BDbiosciences) for 30 minutes. Bone marrow cells were obtained by flushing both tibias as described [21 (link)] and cell surface stained with anti-human CD45, anti-human CD71, anti-human CD235, and anti-mouse Ter-119 antibodies (BDbiosciences). Apoptosis in bone marrow cells was measured using Annexin V/7-ADD apoptosis detection kit (BD Biosciences).
4
Mouse Stomach Cell Immunophenotyping
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The mouse stomach cells were blocked with 2 μL/106 cells of Fc Block (BD) in 100 μL fluorescence-activated cell sorter (FACS) buffer (0.5% FBS, 1 mmol/L EDTA, 0.05% NaN3 in PBS) for 10 minutes at 4°C, followed by washing with FACS buffer to remove Fc Block residue. Cell surface staining was performed in FACS buffer containing antibody cocktails (PerCP [Peridinin chlorophyll] /Cy [Cyanine] 5.5-CD45, APC [Allophycocyanin]/Cy7-CD11b, and APC-F4/80; PerCP/Cy5.5-CD45, APC/Cy7-CD11b, APC-F4/80, Pacific Blue–MHCII, and PE/Cy7-CD93; PerCP/Cy5.5-F4/80, APC/Cy7-CD11b, Pacific Blue–MHCII, PE/Cy7-CD93, APC-CD64, APC-CD21/35, APC-CD204, APC-CD14, and APC-VSIG4) on ice for 1 hour. After washing with FACS buffer 3 times, the cells were subjected to flow cytometry with a BD LSR Fortessa cytometer and data were analyzed with FlowJo software.
For intracellular staining, the Fc-blocked cell surface was stained with antibody cocktails on ice for 1 hour. After washing with FACS buffer, the cells were fixed using Cytofix/Cytoperm solution (BD) for 20 minutes on ice, followed by washing with Perm/Wash solution (BD). Intracellular staining was performed using PE-Il1β (BioLegend) for 1 hour on ice. The cells were washed again with Perm/Wash solution twice and analyzed by flow cytometry using a BD LSR Fortessa cytometer.
For intracellular staining, the Fc-blocked cell surface was stained with antibody cocktails on ice for 1 hour. After washing with FACS buffer, the cells were fixed using Cytofix/Cytoperm solution (BD) for 20 minutes on ice, followed by washing with Perm/Wash solution (BD). Intracellular staining was performed using PE-Il1β (BioLegend) for 1 hour on ice. The cells were washed again with Perm/Wash solution twice and analyzed by flow cytometry using a BD LSR Fortessa cytometer.
5
Immune Cell Profiling in Chronic Cutaneous DTHR
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The spleens and dLNs from wild-type, gp91phox-/-, MPO-/- or iNOS-/- mice with chronic cutaneous DTHR were isolated 24 h after the 5th TNCB ear challenge, pressed through a 70 µm cell strainer to obtain a single cell suspension and washed in PBS with 1 % fetal bovine serum. Additionally, the erythrocytes in the cell homogenates from the spleen were lysed using ACK Lysing Buffer (BioWhittaker, Basel, Switzerland). The cells were counted with the C-Chip Disposable Counting Chamber (NanoEnTek, Seoul, Korea), and 5 x 106 cells per sample were used for staining. The single cell suspensions were stained using the following mAbs: T cell panel: ZombieNIR-Viability, PE-CD45.2, AF700-CD8, FITC-CD3, BV510-CD44, BV650-CD69, BV711-PD-1, BV785-CD127, PE/Cy7-CD62L, PerCP-CD4, BV421-CD25 and FC-Block (BD Biosciences, Franklin Lakes, USA); myeloid cell panel: PE/Cy7-Ly6cC, ZombieNIR-Viability, BV-605-PD-L1, FITC-Ly6G, PerCP-MHCII, APC-CD11c, BV510-CD11b, BV650-CD69, BV785-B220, BV421-CD3, PE-NKp46, and FC-Block (BD Biosciences). Single-cell suspensions were measured on a BD LSR Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (FlowJo, Ashland, USA). The gating strategy for the T cells and myeloid cells is represented in Figure S2 .
6
Visualizing T Cell Synapse Fas Localization
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To visualize Fas localization to the T cell synapse, transduced T cells and FBL tumor cells were combined at a 10:1 ratio, and conjugates were formed by centrifugation (200×g), followed by incubation at 37°C for 30 min. 30 µl of cell suspension was added to an Ibidi μ-Slide VI0.4 (80606, Ibidi) microscopy slide and incubated at 37°C for 15 min. Cell conjugates were fixed and stained in PBS following the Ibidi staining protocol with Fc Block (553142, BD PharMingen), anti-mouse Fas APC (563647, BD Biosciences), and anti-mouse FasL PE (106805, Biolegend).
To determine mitochondria concentration, MitoTracker Deep Red (M22426, Invitrogen) was resuspended in serum-free RPMI (11875–093, Gibco) to a final concentration of 10 nM (1:100,000) and warmed to 37°C before cells were resuspended in the solution at 106 cells/ml and incubated at 37°C for 30 min. Cells were resuspended in PBS and stained in Ibidi μ-Slide VI0.4 (80606, Ibidi) as described above, with Fc Block (553142, BD PharMingen), Hoechst (33342, ThermoFisher), and cholera toxin subunit B AF594 (C34777, Life Technologies). All fluorescent microscopy was performed using a DeltaVision Elite high-resolution microscope (GE Healthcare Life Sciences). Captured images were analyzed with ImageJ (National Institutes of Health).
To determine mitochondria concentration, MitoTracker Deep Red (M22426, Invitrogen) was resuspended in serum-free RPMI (11875–093, Gibco) to a final concentration of 10 nM (1:100,000) and warmed to 37°C before cells were resuspended in the solution at 106 cells/ml and incubated at 37°C for 30 min. Cells were resuspended in PBS and stained in Ibidi μ-Slide VI0.4 (80606, Ibidi) as described above, with Fc Block (553142, BD PharMingen), Hoechst (33342, ThermoFisher), and cholera toxin subunit B AF594 (C34777, Life Technologies). All fluorescent microscopy was performed using a DeltaVision Elite high-resolution microscope (GE Healthcare Life Sciences). Captured images were analyzed with ImageJ (National Institutes of Health).
7
Isolation and Sorting of Lung Immune Cells
8
Monocyte Phenotype Characterization
9
Flow Cytometry for Viable Immune Cells
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Mitotracker Deep Red-stained OT1 LN cells were incubated with eBioScience eFluor780 fixable viability dye (Invitrogen, Thermofisher Scientific) for live/dead cell discrimination, and washed. After blocking with FcBlock at 5 μg/ml for 15 min at 4 °C (Becton Dickinson, San Jose, CA, USA), cells were stained with anti-CD8β conjugated to phycoerythrin (PE) at 2 μg/ml for 15 min at 4 °C (clone YTS156.7.7, Biolegend, San Diego, CA, USA).
10
Bone Marrow Immune Cell Profiling
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Bone marrow cells were flushed and collected from femurs (n = 8 to 10 per group). An amount of 1 × 106 cells was plated per well in a 96-well plate. The staining was performed as described by Collins et al. (43 (link)). Briefly, cells were blocked with Fc block (Becton Dickinson Pharmingen, Franklin Lakes, NJ) for 15 min and then stained with antibodies to CD3 (catalog number 56-0032; eBioscience, San Diego, CA), CD4 (catalog number 11-0041; eBioscience, San Diego, CA), CD8a (catalog number 35-0081; eBioscience, San Diego, CA), GR1/Lys-6G (catalog number 53-5931; eBioscience, San Diego, CA), and CD11b (catalog number 12-0113; eBioscience, San Diego, CA) for 30 min. Then, cells were fixed and data were acquired using the Becton Dickinson LSRII flow cytometer. Data analysis was performed using the FlowJo software package (FlowJo, LLC).
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